Summary of Work: During the past year, studies on HIV protease were aimed primarily at determining the conformational selectivity of proteolytic cleavage, and its consequences. In particular, three of the eight cleavage sites on the Gag-Pol polyprotein which are the target sequences for HIV protease involve Araa-Pro bonds, where Araa corresponds to the aromatic amino acids tyrosine or phenylalanine. Since imide bonds formed with proline exhibit significant cis/trans isomerism, this leads to a question concerning the specificity of cleavage of the Araa-Pro bonds. This specificity can be determined under conditions in which the rate of cleavage greatly exceeds the isomerization rate - i.e., high enzyme concentrations and low temperature. In order to study the reaction, we have utilized two approaches: 1. A fluorogenic substrate peptide analog of the p17/p24 cleavage site of the gag polyprotein with the sequence: Arg-Glu(EDANS)-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-Lys(DABCYL)- Arg was used for HIV protease assays. Hydrolysis of the Tyr-Pro bond results in increased separation of the DABCYL fluorophore form the EDANS quencher, leading to an increase in fluorescence. 2. A fluorinated substrate: Ser-Gln-Asn-FPhe-Pro-Ile-Val-Gln, where FPhe = L-4- fluorophenylalanine, was used for NMR studies. The fluorine nucleus acts as a useful reporter group for both cis/trans isomerization and for cleavage, and in fact provides distinct signals depending on where the peptide is cleaved. As of this date, the 19F NMR studies were limited to the model aspartyl protease, pepsin, since HIV protease proved too unstable for use in the NMR experiments. Specificity for cleavage of the trans imide bond conformation was demonstrated in all cases. The conformational selectivity of proteolytic cleavage of Araa-Pro imide bonds by HIV protease provides a possible explanation for the accumulation of the cellular protein cyclophilin by HIV. In particular, the cyclophilin could act as an auxiliary enzyme for the protease, converting inactive, cis imide bonds into trans bonds which are substrates for the protease.